Francisella tularensis is a highly efficient zoonotic pathogen that is able to establish a virulent infection from 10 organisms or less. To accomplish this feat the bacterium must have devised efficient means for infecting its host, and effective ways of avoiding host defense mechanisms. A few virulence factors, or virulence-associated genes, have been identified, but for the most part their roles have not been well defined. To identify novel virulence factors in F. tularensis, a transposon-mutagenesis library of the Schu S4 strain of F. tularensis tularensis was constructed, and screened for mutants defective in intracellular growth in the hepatic cell line HepG2. This screen identified a mutant in locus FTT1103, which is predicted to encode a hypothetical lipoprotein. In addition to being required for intracellular survival, this locus was also essential for in vivo virulence. The primary hypothesis is that FTT1103 protein represents a novel essential virulence factor. Although the primary amino acid sequence is not closely related to any known protein it does share some sequence similarity to DsbA-Com1 like proteins. Com1 is an outer membrane-associated lipoprotein of Coxiella burnetii. Com1-like proteins are present in a number of gram-negative pathogens but their role as virulence factors has not been explored. Therefore, understanding the role that FTT1103 protein plays in Francisella virulence may potentially have broader implications for the pathogenicity of other bacteria. The overall goal of this proposal is to characterize the essential role of the FTT1103 protein in virulence. This will be approached by examining the protein's subcellular localization and membrane topology, determining what aspects of the protein's primary sequence are critical for its structure and function, and exploring the potential roles this protein plays in virulence.